The carotenoid beta-carotene is a pigment useful in enhancing the pigmentation of animal foodstuffs, food products and cosmetics. Typically, beta-carotene provides yellow to yellow-orange pigmentation. Beta-carotene also serves as a precursor of Vitamin A (retinol) in both animals and man. In addition, beta-carotene, like some other carotenoids, is an effective antioxidant. Epidemiological studies suggest the use of beta-carotene to prevent or treat certain types of cancer and to reduce cellular or tissue damage caused by reactive oxygen species and phototoxic molecules, as occurs, for example, in cardiovascular disease. Beta-carotene may also be used to stabilize compounds subject to oxidation, particularly when exposed to light.
Due to Food and Drug Administration regulations covering chemically-synthesized products, it is preferable to use biological sources to produce beta-carotene. The carotenoid is known to be synthesized by most green plants as well as by certain algae (e.g., Dunaliella), fungi (e.g., Ascomycetes and Deuteromycetes), cyanobacteria and photosynthetic bacteria. Naturally-occurring Zygomycetes of the order Mucorales, family Choanephoraceae, which includes the genera Blakeslea, Choanephora, Mucor, Parasitella, Phycomyces, and Pilaria are particularly well known producers of beta-carotene. Accumulation of beta-carotene in these fungi is strongly linked to sexual interaction between plus mating type (positive) and minus mating type (negative) microorganisms of such fungi. In one example using wild-type Blakeslea trispora, a negative microorganism alone produced 108 milligrams of beta-carotene per liter of medium while a mated culture, formed from the same negative microorganism and a positive microorganism, produced 350 milligrams of beta-carotene per liter of medium (U.S. Pat. No. 3,522,146 by Jager, issued Jul. 28, 1970). In a second example using Phycomyces, a negative microorganism alone produced 5.6 milligrams of beta-carotene per gram dry cell weight while a mated culture, formed between the negative microorganism and a positive microorganism, produced 25 milligrams of beta-carotene per gram dry cell weight (U.S. Pat. No. 4,318,987 by Murillo Araujo et al., issued Mar. 9, 1982).
Investigators have shown that beta-factor, a hormone-like substance that is produced upon mating, stimulates beta-carotene production in Blakeslea. The major component of beta-factor is trisporic acid. Other chemicals that stimulate beta-carotene production include beta-ionone, retinol, kerosene, aromatics (such as dimethyl phthalate and veratrol), and nitrogenous heterocyclic compounds (such as isoniazid and iproniazid). However, past efforts to produce beta-carotene have focussed primarily on improving fermentation conditions rather than on the use of genetic selection techniques to improve beta-carotene production.